Evaluation of a real-time quantitative PCR to measure the wild Plasmodium falciparum infectivity rate in salivary glands of Anopheles gambiae

Alexandra Marie, Anne Boissière, Majoline Tchioffo Tsapi, Anne Poinsignon, Parfait H Awono-Ambéné, Isabelle Morlais, Franck Remoue, Sylvie Cornelie, Alexandra Marie, Anne Boissière, Majoline Tchioffo Tsapi, Anne Poinsignon, Parfait H Awono-Ambéné, Isabelle Morlais, Franck Remoue, Sylvie Cornelie

Abstract

Background: Evaluation of malaria sporozoite rates in the salivary glands of Anopheles gambiae is essential for estimating the number of infective mosquitoes, and consequently, the entomological inoculation rate (EIR). EIR is a key indicator for evaluating the risk of malaria transmission. Although the enzyme-linked immunosorbent assay specific for detecting the circumsporozoite protein (CSP-ELISA) is routinely used in the field, it presents several limitations. A multiplex PCR can also be used to detect the four species of Plasmodium in salivary glands. The aim of this study was to evaluate the efficacy of a real-time quantitative PCR in detecting and quantifying wild Plasmodium falciparum in the salivary glands of An. gambiae.

Methods: Anopheles gambiae (n=364) were experimentally infected with blood from P. falciparum gametocyte carriers, and P. falciparum in the sporozoite stage were detected in salivary glands by using a real-time quantitative PCR (qPCR) assay. The sensitivity and specificity of this qPCR were compared with the multiplex PCR applied from the Padley method. CSP-ELISA was also performed on carcasses of the same mosquitoes.

Results: The prevalence of P. falciparum and the intensity of infection were evaluated using qPCR. This method had a limit of detection of six sporozoites per μL based on standard curves. The number of P. falciparum genomes in the salivary gland samples reached 9,262 parasites/μL (mean: 254.5; 95% CI: 163.5-345.6). The qPCR showed a similar sensitivity (100%) and a high specificity (60%) compared to the multiplex PCR. The agreement between the two methods was "substantial" (κ = 0.63, P <0.05). The number of P. falciparum-positive mosquitoes evaluated with the qPCR (76%), multiplex PCR (59%), and CSP-ELISA (83%) was significantly different (P <0.005).

Conclusions: The qPCR assay can be used to detect P. falciparum in salivary glands of An. gambiae. The qPCR is highly sensitive and is more specific than multiplex PCR, allowing an accurate measure of infective An. gambiae. The results also showed that the CSP-ELISA overestimates the sporozoite rate, detecting sporozoites in the haemolymph in addition to the salivary glands.

Figures

Figure 1
Figure 1
Standard curve of qPCR using serial dilutions of DNA from cultured parasites. Calibration curve was generated using 53 calibrations curves. The curve is based on the known DNA concentration (genomes/μL) and shows the reproducibility. Error bars show the standard deviation for each DNA standard from 6 to 60, 000 genomes/μL [11]. © Boissiere et al[11].

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